CN111568893A - Docetaxel-resveratrol-co-loaded nano long-circulating liposome as well as preparation method and application thereof - Google Patents

Abstract

The invention provides a docetaxel-resveratrol co-carried nano long-circulating liposome as well as a preparation method and application thereof, belonging to the technical field of medicament preparation. The nano long-circulating liposome comprises a medicinal active ingredient and a lipid membrane material, wherein the medicinal active ingredient is docetaxel and resveratrol; the lipid membrane material comprises soybean lecithin, cholesterol and DSPE-PEG 2000. The invention verifies that the traditional Chinese medicine resveratrol with antitumor activity and docetaxel combined have stronger synergistic antitumor effect when acting on human prostate cancer cells and human lung cancer cells for the first time, and simultaneously prepares the human prostate cancer cells and the human lung cancer cells into the nano long-circulating liposome, thereby not only effectively improving the solubility of the long-circulating liposome, but also improving the permeation and retention effects of the medicine, reducing the side effect of the long-circulating liposome on the basis of enhancing the antitumor activity of docetaxel, and laying a foundation for the development and the development of novel docetaxel nano preparations.

Description

A kind of co-loaded docetaxel-resveratrol nanometer long-circulating liposome and preparation method thereof law and application

technical field

The invention belongs to the technical field of pharmaceutical preparation, and in particular relates to a docetaxel-resveratrol nanometer long-circulating liposome co-loaded and a preparation method and application thereof.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

系采用非离子表面活性剂Tween-80和乙醇(50:50，v/v)作为混合溶媒。其中Tween-80可以增加细胞膜流动性和膜渗透性，导致严重的超敏反应、体液潴留等不良反应，并且还会造成溶血和胆汁淤积。临床使用

须预服抗组胺剂和糖皮质激素类，这常常给病人带来痛苦，直接影响了多西他赛的使用。因此，开发具有良好的溶解性能且高效低毒的新型多西他赛给药系统是近些年来的研究热点。Docetaxel (DTX) is a taxane anticancer drug obtained by semi-synthesis of the precursor 10-DAB extracted from the needles of Taxus baccata (Europeanyew). agent. Clinically, DTX is widely used in the treatment of various types of cancer. The FDA has approved its application in the treatment of ovarian cancer, breast cancer, non-small cell lung cancer and prostate cancer. DTX is white or off-white powder with poor water solubility. The bioavailability of ordinary oral preparations is only 8%. The in vivo pharmacokinetics are three-compartment model, and the phase half-lives are 4min, 36min and 11.1h respectively. At present, the only clinical application of docetaxel is injection, the trade name is

The nonionic surfactant Tween-80 and ethanol (50:50, v/v) were used as mixed solvents. Among them, Tween-80 can increase cell membrane fluidity and membrane permeability, resulting in severe hypersensitivity reactions, fluid retention and other adverse reactions, and can also cause hemolysis and cholestasis. clinical use

Antihistamines and glucocorticoids must be pre-administered, which often brings pain to patients and directly affects the use of docetaxel. Therefore, the development of a new docetaxel drug delivery system with good solubility, high efficiency and low toxicity is a research hotspot in recent years.

Resveratrol (Res) is an anthraquinone terpenoid (non-flavonoid) polyphenolic compound, which is abundant in grapes, Polygonum cuspidatum and other plants. It has been found that Res has various physiological activities including anticancer, cardiovascular protection, antibacterial, neuroprotection, and immune regulation. At the same time, in recent years, studies have found that the combination of resveratrol with paclitaxel, doxorubicin and other anticancer drugs can enhance the anticancer activity of the latter. Sprouse et al. found that the combination of resveratrol and paclitaxel could enhance the antitumor activity of paclitaxel on breast cancer cells MDA-MB-231 and its drug-resistant cells MDA-MB-231/PacR. Zhao et al. co-encapsulated resveratrol and doxorubicin in PLGA nanoparticles and acted on doxorubicin-resistant breast cancer cells MCF-7/ADR and MDA-MB-231/ADR. The drug can reverse doxorubicin resistance by inhibiting the expression of resistance-related proteins P-gp, MRP-1 and BCRP, thus revealing the clinical potential of resveratrol in combination with other anticancer drugs.

Liposomes are molecularly ordered assemblies formed spontaneously by phospholipids relying on hydrophobic association in water. They are multi-layered vesicle structures, each layer is a lipid bimolecular membrane, interlaminar and liposome core. It is the water phase, and the bimolecular membrane is the oil phase. According to the liposome structure, the lipid-soluble drugs can be dispersed in the interlayer of the hydrophobic groups of the phospholipid bilayer, so as to improve the solubility of the poorly soluble drugs. Due to its good biocompatibility and controlled release, liposomes have been used in clinical treatment as a mature low-toxicity and high-efficiency preparation, such as paclitaxel liposomes, doxorubicin liposomes and pirarubic acid hydrochloride. Star liposomes, etc. However, the inventors found that liposomes also have some problems and deficiencies, such as poor stability, easy aggregation and leakage, and at the same time, they are easily cleared by the reticuloendothelial system (RES) after entering the blood, so that the lipid The body cannot reach the target site completely, resulting in poor treatment effect.

SUMMARY OF THE INVENTION

Aiming at the above-mentioned deficiencies of the prior art, the present invention provides a docetaxel-resveratrol nanometer long-circulating liposome and a preparation method and application thereof. The present invention verifies the traditional Chinese medicine resveratrol with antitumor activity for the first time. Alcohol combined with docetaxel has a strong synergistic anti-tumor effect on human prostate cancer cells and human lung cancer cells. At the same time, the two are prepared into nanometer long-circulating liposomes, which not only effectively improves their solubility, but also improves the The drug penetration and retention effects reduce the side effects on the basis of enhancing the antitumor activity of docetaxel, which lays the foundation for the development and research of new docetaxel nano-formulations.

In order to realize the above-mentioned technical purpose, the technical scheme of the present invention is as follows:

The first aspect of the present invention provides the application of docetaxel combined with resveratrol in the preparation of antitumor drugs. In the present invention, it is verified for the first time that traditional Chinese medicine resveratrol with antitumor activity combined with docetaxel has strong synergistic antitumor effect on human prostate cancer cells PC3, DU145 and human lung cancer cell H460.

In a second aspect of the present invention, a nanometer long-circulating liposome is provided, the liposome includes a pharmaceutical active ingredient and a lipid membrane material, wherein the pharmaceutical active ingredient is docetaxel and resveratrol; The lipid membrane material includes soybean lecithin, cholesterol and DSPE-PEG2000.

The third aspect of the present invention provides a method for preparing the above-mentioned nanometer long-circulating liposome, the method comprising using a thin film hydration method to prepare the above-mentioned nanometer long-circulating liposome;

The fourth aspect of the present invention provides the application of the above nanometer long-circulating liposomes in the preparation of antitumor drugs.

In a fifth aspect of the present invention, an anti-tumor drug is provided, the anti-tumor drug comprising the above nanometer long-circulating liposome.

The beneficial technical effects of the above one or more technical solutions:

1) For the first time, it was verified that the traditional Chinese medicine resveratrol with anti-tumor activity combined with docetaxel has a strong synergistic anti-tumor effect on human prostate cancer cells PC3, DU145 and human lung cancer cells H460.

中非离子表面活性剂Tween-80可导致病人出现严重的超敏反应、体液潴留等不良反应。通过将两者制备成纳米长循环脂质体不仅可以提高其溶解性，同时可以提高药物渗透和滞留效应，在增强多西他赛抗肿瘤活性的基础上减少其副作用，为新型多西他赛纳米制剂的开发和研制奠定基础。2) Due to the poor water solubility of docetaxel and resveratrol, and the only clinical injection of docetaxel

The non-ionic surfactant Tween-80 can cause severe hypersensitivity reactions, fluid retention and other adverse reactions in patients. By preparing the two into nano-long circulating liposomes, it can not only improve its solubility, but also improve the drug penetration and retention effects, and reduce its side effects on the basis of enhancing the antitumor activity of docetaxel, which is a new type of docetaxel. Lay the foundation for the development and research of nano-formulations.

3) It was confirmed that co-loaded docetaxel-resveratrol long-circulating nanoliposomes can not only significantly inhibit tumor growth in tumor model mice, but also reduce the side effects of docetaxel and prolong the survival time of mice , to provide more clinical application value and research ideas for the docetaxel nano drug delivery system.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from the provided drawings without any creative effort.

Figure 1 shows the cytotoxicity and CI value determination of different molar ratios of DTX and Res on different cells in the examples of the present invention; wherein, (a) PC3, (b) DU145, (c) H460, (d) A549, (e) ) HepG2, (f) HUH7 (n=3);

Figure 2 shows Annexin V-FITC/PI double staining analysis of different cells in the example of the present invention; wherein, (a) PC3, (b) H460; **p<0.01, *p<0.05, the difference between DTX and DTX+Res There is a significant difference between; ##p<0.01, there is a significant difference between Res and DTX+Res;

Fig. 3 is the transmission electron microscope image and in vitro release curve diagram of DTX/Res-LPNs in the embodiment of the present invention; wherein, (a) is the transmission electron microscope image of DTX/Res-LPNs; (b) is the in vitro release curve of DTX and Res Figure; p<0.05, **p<0.01, there is a significant difference between the DTX-Sol group and the DTX/Res-LPNs group; ##p<0.01, there is a significant difference between the Res-Sol group and the DTX/Res-Sol group significant difference;

Figure 4 shows the cellular uptake of coumarin 6 after PC3 cells were incubated with coumarin 6 solution, coumarin 6-LPNs and coumarin 6 solution+Blank-LPNs at 37°C for 4 hours respectively in the example of the present invention Case (bar means 100μm);

Figure 5 is a comparison diagram of human tumor model mice after intravenous injection of different formulation groups in the embodiment of the present invention, wherein (a) is a tumor photo, (b) is a tumor volume change, (c) is a tumor weight change, (d) ) is the survival curve, (e) is the body weight change. *p<0.05, **p<0.01, significant difference compared to control group; ##p<0.01, significant difference between DTX/Res-Sol and Res-Sol.

Figure 6 is the HE and KI67 staining diagrams of mouse tumors in different formulation administration groups in the example of the present invention.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention.

In a specific embodiment of the present invention, the application of docetaxel combined with resveratrol in the preparation of antitumor drugs is provided. In the present invention, it is verified for the first time that traditional Chinese medicine resveratrol with antitumor activity combined with docetaxel has strong synergistic antitumor effect on human prostate cancer cells PC3, DU145 and human lung cancer cell H460.

Wherein, the molar ratio of docetaxel to resveratrol is 1:0.25-4, such as 1:0.25, 1:0.5, 1:1, 1:2 and 1:4.

中非离子表面活性剂Tween-80可导致病人出现严重的超敏反应、体液潴留等不良反应。经研究发现，将多西他赛与白藜芦醇制备成纳米长循环脂质体不仅可以提高其溶解性，同时可以提高药物渗透和滞留效应，在增强多西他赛抗肿瘤活性的基础上减少其副作用。Due to the poor water solubility of docetaxel and resveratrol, and the only clinical injection of docetaxel

The non-ionic surfactant Tween-80 can cause severe hypersensitivity reactions, fluid retention and other adverse reactions in patients. It has been found that the preparation of docetaxel and resveratrol into nano-long circulating liposomes can not only improve its solubility, but also improve drug penetration and retention effects. On the basis of enhancing the anti-tumor activity of docetaxel reduce its side effects.

Therefore, in another specific embodiment of the present invention, a nanometer long-circulating liposome is provided, and the liposome includes a pharmaceutical active ingredient and a lipid membrane material, wherein the pharmaceutical active ingredient is docetaxel and white vera. Atrol; the lipid membrane material includes soybean lecithin, cholesterol and DSPE-PEG2000.

In another specific embodiment of the present invention, the molar ratio of docetaxel to resveratrol is 1:0.25-4, such as 1:0.25, 1:0.5, 1:1, 1:2 and 1: 4. Preferably, the molar ratio of docetaxel and resveratrol is 1:1.

The molar ratio of docetaxel, soybean lecithin and cholesterol is 1:1:1-3, preferably: 1:1:2.

The added amount of DSPE-PEG2000 is 1%-5% (w/w) of the total material.

In another specific embodiment of the present invention, there is provided a method for preparing the above-mentioned nanometer long-circulating liposome, the method comprising preparing the above-mentioned nanometer long-circulating liposome by a thin film hydration method;

Specifically, the preparation method includes:

Co-dissolving the above-mentioned active pharmaceutical ingredients and lipid film material in an organic solvent, evaporating under reduced pressure to form a lipid film, and then drying to completely remove the solvent;

The dried film was hydrated with PBS and then ultrasonically treated to obtain crude liposomes; the crude liposomes were obtained after filtering and granulating.

In another specific embodiment of the present invention, the organic solvent can be selected from any one or more of methanol, absolute ethanol, acetonitrile and dichloromethane;

In yet another specific embodiment of the present invention, the decompression evaporation temperature is controlled to be 30-60°C;

In another specific embodiment of the present invention, the ultrasonic treatment control time is 10-30 minutes;

In another specific embodiment of the present invention, the specific method of filtering and granulating is as follows: passing the crude liposomes through 0.45 μm and 0.22 μm filter membranes in sequence.

In another specific embodiment of the present invention, the application of the above nanometer long-circulating liposomes in the preparation of antitumor drugs is provided.

In yet another specific embodiment of the present invention, an anti-tumor drug is provided, and the anti-tumor drug includes the above-mentioned nanometer long-circulating liposome.

The anti-tumor drug provided by the present invention can also be prepared into different dosage forms by adding suitable excipients and additives and using techniques known in the art, such as injection, lyophilized powder for injection, liposome suspension Liquid, tablet, gel, implant, etc., for intravenous injection, local injection, implantation, inhalation and other routes of administration.

It should be noted that tumors are used in the present invention as known by those skilled in the art. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in the body. Conversely, malignancies are defined as cells with a variety of cellular and biochemical abnormalities capable of developing systemic disease (eg, tumor metastases in distant organs).

In another specific embodiment of the present invention, the medicament of the present invention can be used to treat malignant tumors. Examples of malignant tumors that can be treated with the medicaments of the present invention include solid tumors and hematological tumors. Solid tumors can be, for example, breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (eg, thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, Tumors of the larynx and hypopharynx, mesothelioma, tumors of the ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testes, stomach, skin, ureter, vagina, and vulva. Malignancies include hereditary cancers such as retinoblastoma and Wilmstumor. In addition, malignant tumors include primary tumors in said organs and corresponding secondary tumors (tumor metastases) in distant organs. Hematological tumors can be, for example, aggressive and painless forms of leukemias and lymphomas, namely non-Hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), acute lymphoblastic leukemia (ALL), Hodgkin's disease, Multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, tumor-like syndromes, and cancers of unknown primary site and AIDS-related malignancies;

In yet another specific embodiment of the present invention, the medicament of the present invention can be used for the treatment of prostate cancer and lung cancer, especially prostate cancer.

It should be noted that malignancies do not necessarily require the formation of metastases in distant organs. Certain tumors exert devastating effects on the primary organ itself through their aggressive growth properties. These can lead to structural destruction of tissues and organs, ultimately leading to failure and death of the indicated organ.

The present invention is further explained and illustrated by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention.

Example

1. Research process

1) Study on the anti-tumor effect of different proportions of docetaxel-resveratrol combined

Different ratios of docetaxel-resveratrol were applied to human prostate cancer cells (PC3, DU145), lung cancer cells (A549, H460) and liver cancer cells (HepG2, HUH7) to study resveratrol by inhibiting proliferation and inducing apoptosis The synergistic anti-tumor activity of atrol and docetaxel was evaluated by the Combination index (CI) to evaluate the combined anti-tumor effect of the combination regimen based on different ratios of DTX-Res on tumor cells. Calculated as follows:

Among them, D _m1 refers to the corresponding concentration when Drug-1 alone produces x cell inhibition rate; D ₁ refers to the corresponding Drug-1 when combined with Drug-2 to produce the same x inhibition rate 1 concentration; for the same reason, D _m2 refers to the concentration corresponding to the inhibition rate of x cells produced by single drug use, and D ₂ refers to the corresponding Drug- 2 concentrations.

2) Preparation and quality evaluation of co-loaded docetaxel-resveratrol long-circulating nanoliposomes

In this study, the traditional Chinese medicine resveratrol with antitumor activity was used in combination with docetaxel for the first time. Soy lecithin, cholesterol and DSPE-PEG2000 were used as the main membrane materials to prepare co-loaded docetaxel-white membrane by thin film hydration method. Veratrol long-circulating nanoliposomes. 1 mol of docetaxel, 1 mol of resveratrol, 1 mol of soybean lecithin, 2 mol of cholesterol were dissolved in 20 mL of dichloromethane together with 2% DSPE-MPEG2000 (w/w). The mixture solution was evaporated under reduced pressure at 50°C to form a lipid film, and then vacuum dried to completely remove the solvent. The dried membrane was hydrated with PBS (pH 7.4) followed by sonication for 20 minutes to obtain crude liposomes. The crude liposomes were granulated through 0.45 μm and 0.22 μm filters to obtain the final desired docetaxel-resveratrol long-circulating nanoliposomes. The same method was used to prepare docetaxel-encapsulated long-circulating nanoliposomes (DTX-LPNs), resveratrol-encapsulated long-circulating nanoliposomes (Res-LPNs), and coumarin-6-encapsulated nanoliposomes. Long-circulating nanoliposomes (Coumarin6-LPNs), and drug-free long-circulating nanoliposomes (Blank-LPNs).

3) To study the in vitro release and cellular uptake of co-drug-loaded liposomes

a. In vitro release behavior:

The release behavior of co-loaded docetaxel-resveratrol long-circulating nanoliposomes was investigated in PBS buffer solution at pH 7.4 (simulating a humoral environment). The release medium was taken at different time points for high performance liquid chromatography analysis and determination, and the release curve was drawn by the cumulative release percentage (%) of the drug against time;

b. Cell uptake behavior:

Coumarin-6-loaded long-circulating nanoliposomes were prepared, the concentration of coumarin-6 was 10 μg/mL, and the negative control group was 10 μg/mL coumarin-6 solution. Take the logarithmically proliferated PC3 cells, digest with trypsin, resuspend by centrifugation, count in a cell counter, inoculate a 6-well plate at a density of 2×10 ⁵ cells/well, and incubate in the incubator until the cells adhere to the wall, then remove the culture medium. The experimental group and the control group were added to the preparations. After culturing for 4 h, the solution in the well was discarded, and the pre-cooled (4°C) PBS solution was washed three times to remove residual free fluorescent substances, and 0.5 mL/well PBS was added to infiltrate the cells. The treated 6-well plate was placed under a confocal microscope to observe the fluorescence intensity of each well and photographed.

2) To study the in vivo antitumor activity of co-drug-loaded liposomes and the evaluation of their side effects (key improvement points)

a. Evaluation of antitumor activity:

6-week-old male Balb/c nude mice were injected subcutaneously with PC3 cells (2×10 ⁵ cells/0.1 mL) in the right hind limb. When the tumor volume was ≥100 mm, the mice ^were randomly divided into six groups (five mice in each group): (1) 0.9% saline group (Control); (2) docetaxel aqueous solution group (DTX-Sol ); (3) Resveratrol aqueous solution group (Res-Sol); (4) Docetaxel/Resveratrol mixed aqueous solution group (DTX/Res-Sol); (5) Docetaxel-Resveratrol Atrol long-circulating nanoliposome group (DTX/Res-LPNs); (6) blank long-circulating nanoliposome group (Blank-LPNs). Dosed by tail vein injection every three days, the dose of DTX was 20 mg/kg and the dose of Res was 11.3 mg/kg (based on a 1:2 molar ratio of DTX/Res). The body weight and tumor size of the mice were determined during the treatment. Tumor volume was measured by vernier calipers and calculated using the following equation:

V=a×b ² /2

where a and b represent the longest and shortest diameters of the tumor, respectively. Immediately after the mice were sacrificed on day 48, tumor tissue was harvested, cut into 5 μm thick sections, and stained with H&E (hematoxylin and eosin) to observe histopathological changes.

2. Research results

1) Resveratrol combined with docetaxel has a strong synergistic anti-tumor effect on human prostate cancer cells PC3, DU145 and human lung cancer cells H460

Cell survival was evaluated after incubating different cells with docetaxel and resveratrol in different molar ratios for 48 hours. By calculating the CI value as an indicator of response synergy. CI values less than, equal to and greater than 1 indicate synergy, additive effect and antagonism, respectively. The closer the CI value is to 0, the stronger the synergy. As shown in Figures 1a, 1b and 1c, the combination of Res and DTX showed higher cytotoxicity in PC3, DU145 and H460 cells compared with DTX and Res alone, suggesting that the combination therapy has a greater application potential. Even for A549, HepG2 and HUH7 cells (Figures 1d, 1e and 1f), the combination at certain molar ratios showed enhanced cytotoxicity compared to either drug alone. It can be seen from Figure 1 that the molar ratio of DTX and Res showed the best anti-tumor effect in PC3, DU145 and HepG2 cells when the molar ratio was 1: 2; the optimal anti-tumor effect was shown in A549 cells when the molar ratio was 1: 1; When the molar ratio was 1:4, it showed the best anti-tumor effect in H460 cells; when the molar ratio was 4:1, it showed the best anti-tumor effect in HUH7 cells.

2) The combination of resveratrol and docetaxel can significantly induce the apoptosis of H460 and PC3 cells

As shown in Figure 2, for PC3 cells, the percentage of apoptotic cells (the sum of early apoptotic and late apoptotic cells) of 20μM DTX+40μM Res combination was 22.99±1.17%, which was significantly higher than 20μM DTX (14.59±3.01%). %, p<0.05) or 40 μM Res (11.29±0.80%, p<0.05) administered alone. In addition, the percentage of apoptotic cells in the combined administration of 40μM DTX+80μM Res was 69.60±5.94%, which was also significantly higher than that of 40μM DTX (35.65±0.92%, p<0.01) or 80μM Res (22.27±3.87%, p<0.01) ) alone (Fig. 2a). For H460 cells, the percentage of apoptotic cells in the combination of 20μM DTX+40μM Res was 39.29±0.19%, which was significantly higher than that of 20μM DTX (17.89±0.39%, p<0.01). (Fig. 2b).

3) Characterization of co-loaded docetaxel-resveratrol long-circulating nanoliposomes

As shown in Table 2, the average particle size of different types of liposomes ranged from 80 to 110 nm, among which the particle size of DTX/Res-LPNs was 99.76 nm, and all liposomes were negatively charged. The encapsulation efficiencies of DTX and Res in DTX/Res-LPNs were 81.52±3.13% and 85.27±1.74%, respectively. Transmission electron microscope observation showed that the DTX/Res-LPNs were multi-layered structures and nearly spherical in shape. (Fig. 3a).

Table 1. Characterization of different types of liposomes (NA means none)

As shown in Figure 3b, in phosphate buffer at pH 7.4, the in vitro release of docetaxel and resveratrol showed a burst phenomenon in the first 4 hours, and the cumulative release of DTX exceeded 70%, while The cumulative release of Res is close to 60%. But for the co-loaded docetaxel-resveratrol long-circulating nanoliposome group, in the first 12 hours, the cumulative release of DTX only reached 48%, and the cumulative release of Res only reached 37% , the results show that the co-drug-loaded liposomes have good controlled release ability.

In order to investigate the cellular uptake ability of nanoliposomes by PC3 cells, long-circulating nanoliposomes encapsulated with fluorescent dye coumarin-6 were selected to observe their cellular uptake behavior. As shown in Figure 4, when PC3 cells were co-cultured with coumarin 6-LPNs, their green fluorescence was enhanced in the cytoplasm compared with other groups, indicating that liposomes have a good ability to deliver drugs to PC3 cells . In addition, a large amount of green fluorescence was not observed in the mixture group of coumarin 6-solution and blank-LPNs, which proved that the co-delivery efficiency of the simple drug solution and blank liposome mixture was not high.

4) Therapeutic effect of co-loaded docetaxel-resveratrol long-circulating nanoliposomes on prostate cancer

As shown in Figure 5, the tumor volume and tumor weight of the mice in the saline-treated group increased rapidly, while the tumor volume of the mice in the DTX alone, DTX/Res-Sol and DTX/Res-LPNs groups decreased significantly. The mice in the DTX/Res-LPNs group significantly inhibited tumor growth (p<0.05), which was significantly different from the free Res group. It should be noted that mice in the DTX alone and DTX/Res-Sol groups were significantly inhibited in tumor volume growth during the first 7 days post-dose, but all mice in these two groups experienced significant inhibition of tumor volume growth on days 9 and 9, respectively. 8 days of death (Fig. 5d). The likely reason is that high doses of DTX not only inhibited tumor growth during treatment, but also produced strong toxicity and side effects in mice. However, the liposome group reduced the toxicity and side effects to the mice by releasing the drug slowly, so there was no premature death (Fig. 5b).

The safety of the formulation was evaluated by the survival curve and body weight of the mice. As shown in Figures 5d and 5e, compared with the DTX/Res-LPNs group, the mice in the DTX solution alone and DTX/Res mixed solution group showed severe weight loss, but the mice in the liposome group did not. Significant weight loss. And the mice in the liposome group did not die until 48 days after administration, which indicated that the liposome group had higher safety than other formulation groups.

As shown in Figure 6, co-loaded docetaxel-resveratrol long-circulating nanoliposomes induced nucleolysis and apoptosis in a large number of tumor cells compared with other formulation groups, confirming that DTX/Res-LPNs Can effectively inhibit tumor growth.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention but not to limit them. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or equivalently replace the technical solutions of the present invention as required without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. The docetaxel and resveratrol combined application in preparing antitumor drugs.

2. The use of claim 1, wherein the molar ratio of docetaxel to resveratrol is 1: 0.25-4, preferably 1: 0.25, 1: 0.5, 1: 1. 1: 2 and 1: 4.

3. a nanometer long-circulating liposome is characterized in that the liposome comprises a pharmaceutical active ingredient and a lipid membrane material, wherein the pharmaceutical active ingredient is docetaxel and resveratrol; the lipid membrane material comprises soybean lecithin, cholesterol and DSPE-PEG 2000.

4. The nanovesicle liposomes of claim 3, wherein the molar ratio of docetaxel to resveratrol is 1: 0.25-4, preferably 1: 0.25, 1: 0.5, 1: 1. 1: 2 and 1: 4.

5. the Nanotrycirculating liposome of claim 3, wherein the molar ratio of docetaxel, soy lecithin and cholesterol is 1: 1: 1-3, preferably 1: 1: 2;

the adding amount of the DSPE-PEG2000 is 1 to 5 percent of the total material, and the w/w ratio is higher.

6. The method for producing a nano-sized long-circulating liposome according to any one of claims 3 to 5, wherein the method comprises producing the nano-sized long-circulating liposome by a thin film hydration method.

7. The method of claim 6, comprising:

co-dissolving the pharmaceutically active ingredient and lipid membrane material in an organic solvent, evaporating under reduced pressure to form a lipid film, and drying to completely remove the solvent;

hydrating the dried film with PBS and then carrying out ultrasonic treatment to obtain a crude liposome; filtering and grading the coarse liposome to obtain the final product.

8. The method according to claim 6, wherein the organic solvent is selected from one or more of methanol, absolute ethanol, acetonitrile and dichloromethane;

the reduced pressure evaporation temperature is controlled to be 30-60 ℃;

the ultrasonic treatment control time is 10-30 minutes;

the specific method for filtering and straightening the grains comprises the following steps: the crude liposomes were passed through 0.45 μm and 0.22 μm filters in sequence.

9. Use of the nano-sized long-circulating liposome of any one of claims 3 to 5 for the preparation of an antitumor drug; preferably, the tumors are prostate cancer and lung cancer.

10. An antitumor agent comprising the nano-sized circulating liposome according to any one of claims 3 to 5.

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